Interleukin 12, Human (IL-12), BioAssay(TM) ELISpot Kit

Intended Use:\nThis 2.5 hour ELISpot kit is developed to detect and visualize of single cells secreting human IL-12.\n\nPrinciple:\nAdd stimulated cells or cells and stimulant to the wells and incubate at 37 degrees C in CO2 incubator for a specified period. Secreted human IL-12 binds to antibody coated microtiter plate.\nmicrotiter plate coated with human IL-12 Capture Antibody. Cells and unbounded proteins are washed away. Streptavidin-AP or Streptavidin-HRP is added and binds to the biotinylated detection antibody. Unbounded proteins are washed away. Biotinylated detection antibody is added and binds to the secreted human IL-12. Substrate Solution is added. A colored precipitate forms and appears as spots at the sites of human IL-12 secreting location. Each individual spot representing an individual human IL-12 secreting cell.\n\nBackground:\nInterleukin 12 (IL-12), also known as natural killer cell stimulatory factor (NKSF) and cytotoxic lymphocyte maturation factor (CLMF), is a pleitopic cytokine originally identified in the medium of cultured EBV-transformed RPMI-8866 cells) (1-3). IL-12 is a 75kD glycoprotein heterodimer composed of two unequal, genetically-unrelated subunits. The smaller subunit (p35) has homology to IL-6 and G-CSF while the larger subunit (P40) shows recognizable similarity to the soluble receptor for IL-6, leading to the suggestion that IL-12 might have evolved from a cytokine/soluble receptor complex (2-6). Cells known to produce IL-12 include monocytes/macrophages, B cells and connective tissue type mast cells (7-10). IL-12 shows species specificity with human IL-12 reportedly showing minimal activity in the murine system (3,7). For reviews on IL-12, see references (8-12).\n\nEach subunit of IL-12 apparently arises from a single copy gene. The transcription of the subunit of the subunit mRNAs is closely co-ordinated, although an excess of the larger subunit (P40) has been shown to be produced by B cells in addition to active IL-12 (1,3). Expression of the smaller chain (p35) is reported to be enhanced by simultaneous expression of the larger chain (p40). Although IL-12 activity cannot be demonstrated in the absence of either chain, the presence of only p40 is associated with inhibition of IL-12-associated activities (3,4). As suggested by their names, p35 has a native molecular weight of 35 kD while p40 has a native molecular weight of 40 kD. In humans, p35 is 197 amino acid residues in length with a predicted molecular weight of 22.5 kD. The molecule contains 7 cysteine residues plus 3 potential N-glycosylation sites and the molecule is believed to be heavily glycosylated. The p40 subunit is 306 amino acid residues in length with a predicted molecular weight of 34.7kD. The molecule contains 10 cysteine residues and four potential N-linked glycosylation sites (3). The murine p35 subunit shows 60% sequence identity with the corresponding human subunit and is 193 amino acid residues in length with seven conserved cysteines and one possible N-linked glycosylation site. Murine p40 shows 70% sequence identity to human p40 and is 313 amino acid residues in length with eleven conserved cysteines and three potential N-linked glycosylation sites (7). In both human and mouse p35 and p40, the mature molecules separate functions can be attributed to p35 and p40. It is not clear what separate functions can be attributed to p35 and p40. Preliminary evidence suggests however that p40 is involved in receptor binding and p35 is important for signal transduction (13).\n\nA unique high affinity receptor for IL-12 (IL-12R) has been characterized from PHA-stimulated human peripheral blood mononuclear cells (14). Approximately 110 kD as determined by cross-linking studies, it has a Kd in the range of 100-600 pM (14). Cross-linking studies also suggested an association with a second protein of approximately 85 kD. IL-12 receptor has also been reported to be present on PHA or IL-2 stimulated CD4+, CD8+, and CD56+ cells and on one T cell and one NK cell line (14, 15).\n\nIL-12 is produced by macrophages and B lymphocytes and has been shown to have multiple effects on T cells and natural killer (NK) cells (16, 17). These include inducing production of IFN-g and TNF by resting and activated T and NK cells, synergizing with other IFN-g inducers at both the transcriptional and post-transcriptional levels to induce IFN-g gene expression, enhancing the cytotoxic activity of resting NK an d T cells, inducing and synergizing with IL-2 in the generation of lymphokine-activated killer (LAK) cells, acting as a comitogen to stimulate proliferation of resting T cells and inducing proliferation of activated T and NK cells (16). Evidence indicates that IL-12 produced by macrophages in response to infectious agent, is a central mediator of the cell-mediated immune response by its actions on the development, proliferation, and activities of TH1 cells (8,9, 18,19). In its role as the initiator of cell-mediated immunity, it has been suggested that IL-12 has therapeutic potential as a stimulator of cell-mediated immune responses to microbial pathogens, metastatic cancer, and viral infections such as AIDS (8,9, 18-20).\n\nKit Components:\nA. Mictotiter Plates 1x96wells PVDF-bottom Immunospot plates pre-coated with mouse anti-human IL-12 monoclonal antibody. \nB. Positive Control 1x1 vial. Supplied as a Lyophilized recombinant human IL-12. Reconstitute with 250 ul Cell Culture Media before use. Use in 1 hour. \nC. Wash Buffer (20x) 1x60ml Add 1 volume of Wash buffer (20x) to 19 volume of ddH2O. Use in 1 week. Stored at room temperature.\nD. IL-12 (Biotin) 1x11ml Biotinylated mouse anti-human IL-12 monoclonal antibody. Ready to use. \nE. Streptavidin (AP)(100x) 1x120ul. Add 1 volume of Streptavidin (AP)(100x) to 100 volumes of F. (Streptavidin (AP) Diluent) before use. Use in 1 month. Stored at 2-8 degrees C. \nF. Streptavidin (AP) Diluent 1x11ml. Ready to use. \nG. BCIP/NBT Substrate Solution 1x11ml. Ready to use.\n\nStorage and Stability: \nStore other components at 4C. Stable for at least 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.

Prices direct from United States Biological

Quick response times

Exclusive Biosave savings/discounts

SPECIFICATIONS

Catalog Number

I8434-04E

Size

96Tests

Applications

ELISA

Reactivities

Hum

References

1. Kobayahi, M et al. (1989). J. Exp. Med. 170:827.\n2. Stern, A.S et al. (1990) Proc. Natl. Acad. Sci. USA 87:5808.\n3. Gubler, U. et al. (1991). Proc. Natl. Acad. Sci. USA 88:4143\n4. Wolf, S.F et al (1991). J. Immunol. 146:3074.\n5. Merberg, D.M. et al. (1992). Immunol. Today. 13:78\n6. Gearing, D.P. and D. Cosman (1991). Cell 86:9.\n7. Schoendhauf, D.S. et al. (1992). J. Immunol. 148:3433.\n8. Locksley, R.M. (1993). Proc. Natl. Acad. Sci. USA 90:5879.\n9. Trubchleri, G. (1993). Immunol. Today. 14:335.\n10. Reiter, Z. and B. Rappaport. (1993). Crit Rev. Immunol. 13:1.\n11. Brunda, M.J. (1994). J. Leukocyted Biol. 55:280.\n12. Wolf, S.F et al. (1994). Stem Cells 12:154.\n13. Ling, P. et al. (1993). J. Immunol. 150:207A (Abstract)\n14. Chizzonile, R. et al. (1992). J. immunol. 148:3117.\n15. Desal, B.B. et al. (1992).J. Immunol. 148:3125.\n16. D